title: Pyrosequencing analysis of EGFR and KRAS mutations in EUS and EBUS-derived cytologic samples of adenocarcinomas of the lung.
abstract: BACKGROUND
  Patients with stage IV non-small-cell lung cancer harboring an activating epidermal growth factor receptor (EGFR) mutation are eligible for treatment with EGFR tyrosine kinase inhibitors. With pyrosequencing, low-frequency mutations may be detected more easily even in small diagnostic samples like endoscopic ultrasound-guided fine needle aspirations (EUS-FNA) and endobronchial ultrasound-guided transbronchial needle aspirations (EBUS-TBNA). The diagnostic performance of pyrosequencing in analyzing cytological specimens is compared with the routinely used high-resolution melting (HRM) and Sanger sequencing.
  
  
  METHODS
  Patients diagnosed with adenocarcinoma of the lung were selected from a fine needle aspiration and transbronchial needle aspiration specimen database. If formalin-fixed paraffin-embedded tumor blocks were available, mutation analysis was performed for EGFR and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog genes using both pyrosequencing and HRM. When HRM showed abnormalities, Sanger sequencing was used.
  
  
  RESULTS
  A total of 126 samples were available for mutation analysis. The analysis success rate for pyrosequencing and HRM were 97% and 93%, respectively. HRM failures were observed in fragmented DNA showing chains of 100 to 200 bp. A significant correlation between length of DNA fragments (100-300 bp versus 300-400 bp) and mean sample age (797 versus 317 days) was found (p < 0.0001), suggesting an influence of sample age on DNA quality.
  
  
  CONCLUSIONS
  Pyrosequencing on cytological blocks, especially older tumor blocks, is feasible with a high diagnostic success rate. Failures in HRM were observed in DNA samples with short fragments related to longer storage times.
authors.affiliation: Department of Pulmonology, Isala Klinieken, Zwolle, Amsterdam, The Netherlands. j.a.stigt@isala.nl
authors.forename: Jos A
authors.initials: JA
authors.lastname: Stigt
authors.forename: Nils A
authors.initials: NA
authors.lastname: 'tHart
authors.forename: Ageeth J
authors.initials: AJ
authors.lastname: Knol
authors.forename: Steven M
authors.initials: SM
authors.lastname: Uil
authors.forename: Harry J M
authors.initials: HJ
authors.lastname: Groen
mesh_terms: D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000077192:Adenocarcinoma of Lung; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002452:Cell Count; D061765:Endoscopic Ultrasound-Guided Fine Needle Aspiration / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics*; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D017422:Sequence Analysis, DNA / Q000379:methods*; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins